Rheumatoid arthritis (RA) in most populations is highly associated with a set of alleles at the B1 locus of the HLA-DR region (HLA-DRB1 *0101, *0401, *0404, *0405, * 1001, *0408, *1402, *0102 and *1303) called “shared epitope” alleles. The *0401 and *0404 alleles also called DR1 and DR4, respectively, on antigen presenting cells bind to a minimum dominant determinant on type II collagen (CII) residues 263-270 (FKGEQGPK)(SEQ ID NO: 1). Altered peptide ligands (APLs) are peptides that retain enough of the present sequence to be recognized by T cells in the MHC/peptide T cell receptor (TCR) trimolecular complex but interfere with normal signaling through the TCR. APLs can act as antagonists or partial agonists. In animal models, APLs have been shown to be effective in preventing and ameliorating tissue-specific autoimmune diseases. Trials of APL in human autoimmune disease have had mixed results. However, none of these trials incorporated a pre-selection step wherein the APL showed ability in vitro to down regulate Th1 response by patient's peripheral blood mononuclear cells (PBMC) stimulated by an autoantigen as described by herein.
Collagen Autoimmunity in RA
Autoimmunity plays a central role in a diverse group of diseases that afflict man, including rheumatoid arthritis (RA). This systemic disease is characterized by chronic synovitis, which shows a predilection for diarthrodial joints. If unchecked, the synovial inflammation typically produces irreversible joint destruction and permanent disability. A preponderance of evidence indicates that an antigen-driven immune process against one or more proteins found in cartilage sustains synovial inflammation in RA. Although several antigens have been proposed to be involved in the autoimmune response in RA, CII has received the most attention as a candidate autoantigen. It is the predominant protein of articular cartilage, and autoimmunity to CII is common in patients with RA with >60% of RA patients having large amounts of anti-CII cartilage bound antibodies in arthritic joints. Mullazehi and colleagues have reported that autoimmunity to CII correlates with disease severity. RA patients with high serum levels of anti-CII antibodies have a distinct clinical phenotype characterized by an early acute phase response associated with more severe radiological damage in the joints at the time of diagnosis. Moreover, immune complexes from RA synovia, which contained anti-CII antibodies, induced the proinflammatory cytokine tumor necrosis factor (TNF)α and this induction directly correlated with the number of swollen and tender joints. Blockade of the monocyte receptor, Fc gamma RIIa, decreased TNFα production in the joints. These data demonstrate a direct link between the level of autoimmunity to CII and the severity of RA and in such patient's downregulation of autoimmunity to CII should lead to modulation of the destructive arthritis itself.
A form of CII based therapy has been tried in patients with RA and juvenile RA by administering intact native chick or bovine CII to patients. Seven trials of treatment with oral CII have been reported including two of our own. In each case, the native CII was well tolerated with no or mild adverse events. Two of the studies gave favorable results, demonstrating small, but significant, disease improvement. However, the therapeutic window was narrow, suggesting a need to fine-tune future trials of collagen-based therapy. Most patients in these studies also received NSAIDs which block oral tolerance and systemic tolerance. The use of NSAIDS might have been responsible for the mixed results of oral CII in RA. We have conducted studies in murine models of oral tolerance that show the PGE1 analog, misoprostol, reverses NSAID inhibition of oral tolerance and that DMARDs and the anti-TNFα biologic, etanercept, do not block oral tolerance induction. Furthermore, we have applied these crucial data in designing oral tolerance trials in humans. We can down regulate with oral CII, PBMC production of IFNγ to α1(II) in RA patients who take NSAIDS plus misoprostol and/or DMARDS and anti-TNFα therapies. Oral administration of low dose antigen generates regulatory T cells, which act in the respective microenvironment in a non-antigen specific manner by producing down-regulatory cytokines such as IL4, IL10 and TGFbeta, a Th2/Th3 cytokine pattern. Recent studies in mice show that orally administered antigen can induce CD4+ CD25+ Fox P3+ regulatory T cells (Tregs) via retinoic acid dependent mechanisms.
Altered Peptide Ligands in the Treatment of Human Disease
APLs are analogs of determinants recognized by T cells in the MHC/peptide/TCR trimolecular complex. These altered peptides interfere with normal signaling through the TCR. The APL is bound by MHC but because it is presented differently than the naturally occurring epitope, it is recognized aberrantly. APL may act as antagonists or partial agonists. Studies in animal systems have shown that APLs are effective in preventing or ameliorating many tissue-specific autoimmune diseases. Based on the experience in animals, APLs should provide a relatively nontoxic and highly specific therapy for humans with tissue-specific autoimmune diseases. However, human trials have met with mixed results. Treatment of multiple sclerosis with peptide analogs of myelin basic protein administered subcutaneously resulted in an increase in disease activity. On the other hand, trials in type 1 diabetes and in RA have shown promising results. Raz and coworkers treated 35 subjects who had type 1 diabetes with peptide p277, a peptide analog from heat-shock protein hsp60. The treatment group received a total of three injections. After 10 months the treatment group had higher levels of C peptide and reduced need for exogenous insulin as compared to controls. Prakken and coworkers treated 15 patients with RA using a peptide analog of a heat-shock protein, dnaJP1, which shares sequence homology with the shared epitope. Subjects were treated orally with 3 different dosages for six months. Reactivity to dnaJP1 was significantly altered with a decrease in the number of cells producing IFN-γ and IL-2. There was a concomitant increase in IL-4 and IL-10 producing cells. Efficacy in producing a change in the clinical manifestations of disease was not determined due to the small number of patients studied.
We have shown that mice normally resistant to CIA that are transgenic for the human RA MHC susceptibility genes DR1 and DR4 develop arthritis after immunization with human CII. This arthritis can be prevented by administration of an APL that we have named A12. Although the precise mechanism by which the A12 peptide exerts its effect is not yet clear, the interaction of the APL/MHC complex with the TCR appears to play a key role in influencing the differentiation of naive T cells into effector cells.
When optimal engagement occurs between a TCR and a specific antigen in the context of a class II MHC molecule, signal transduction events are initiated. Minor variations in the physicochemical properties of amino acid residues of the peptide which interact with either MHC or TCR can lead to disparate immunological responses. For example, an APL containing a single amino acid substitution at the TCR contact residue of a cytochrome-C peptide has been shown to induce immune deviation to a Th2-type response as compared to the WT agonist peptide, which induced a Th1 response. A double APL based on 2 epitopes in acetylcholine recognized by patients with myasthenia gravis induced generation of CD4+ CD25+ FoxP3+ regulatory T cells and induced Fas dependent and Fas independent apoptosis of acetylcholine-specific T cells. A shift to a Th2 cytokine profile may be significant for the development of arthritis, because Th2 cytokines have inhibitory effects on CIA. IL-10 is effective in inhibiting CIA when administered to mice. IL-4 has a similar effect although it does not duplicate the effects of IL-10.
One of the fundamental and most challenging goals of immunological research is to devise a treatment that suppresses immunity enough to halt an injurious autoimmune process, without disrupting the beneficial functions of the immune system such as surveillance for opportunistic infections and tumors. To this end, analog (or altered) peptide ligands are particularly desirable types of antigen-specific immunotherapy and are well suited for treating autoimmune diseases. They can specifically down-regulate an inflammatory autoimmune response in diarthodial joints where CII is located. Although the use of APLs as therapies for human illness is still in its infancy, recent reports suggest that APLs can modulate autoimmune arthritis by inducing regulatory T cells.
Because RA is strongly associated with DR genes, it seems likely that immune-mediated mechanisms are critically involved, either in the initiation of disease or in its progression to severe joint destruction with systemic manifestations, or both. An antigen that could perpetuate an autoimmune-mediated response is CII. There is incontrovertible evidence that RA is characterized by autoimmunity to CII. Most patients with RA have local production of antibodies to CII in joint tissues. Similar antibodies have been found to be pathogenic in mice with CIA. In mice the autoantibody response is CD4 T cell driven and strongly associated with class II immune response genes including human DR4 and DR1. Other investigators have concluded, “The genetic associations between HLA-DR alleles and antibodies to CII in RA patients is in keeping with the collagen-induced arthritis model and implicates autoimmunity to CII as a major component in the multifactorial pathogenesis of RA”. The ultimate test of that possibility is specifically altering the immune response to CII and determining its affect on disease. Using APLs described herein addresses these issues.